Dna polymerases

ABSTRACT

The present invention provides a DNA polymerase including the sequence of SEQ ID NO. 1 or a sequence which is at least 70% identical thereto, but wherein the aspartic acid residue at position 18 of SEQ ID NO. 1, or the equivalent aspartic acid residue in other sequences, has been replaced by a non-negatively charged amino acid residue. It further provides DNA polymerases comprising the amino acid sequences of SEQ ID NO. 2, 11 and 12 and variants thereof. The present invention also provides nucleic acids encoding the DNA polymerases, a method of producing said DNA polymerases, and compositions, expression vectors and host cells or viruses comprising said DNA polymerases. The present invention also provides uses of said DNA polymerases in nucleotide polymerisation, amplification, and sequencing reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/772,690, filed Jun. 12, 2020, which is a National Stage Applicationof PCT/EP2018/085342, filed on Dec. 17, 2018, which claims the benefitof GB 1721053.5, filed on Dec. 15, 2017, each of which is hereinincorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

This application is being filed electronically via EFS-Web and containsa sequence listing entitled “60_13468_13_ST25_txt” created on Aug. 9,2022, and is 30,804 bytes in size in Computer Readable Form (CRF). Thepaper copy of the sequence listing and the CRF are identical and areincorporated herein by reference.

The present invention relates to DNA polymerases. In particular, thepresent invention relates to modified DNA polymerases with enhancedstrand displacement activity (SDA).

The gold standard of microbial identification still remains culturingand subsequent phenotypic differentiation of the causative agent, aprocess often taking several days to perform and analyze, and this delaymay have major impact on morbidity and mortality of an infectiousdisease. In addition, many organisms cannot grow on culture media,hence, will be undetected by existing culturing methods.

There is a global need to monitor and diagnose critical infectiousdiseases such as HIV/AIDS, tuberculosis, malaria, cholera etc. Thechallenge becomes even more critical in potential epidemic situationssuch as Ebola, avian and swine influenza outbreaks. Despite advances indiagnostic technologies, many patients with suspected infections receiveempiric antimicrobial therapy rather than appropriate therapy dictatedby the rapid identification of the infectious agent. The result isoveruse of our small inventory of effective antimicrobials whose numberscontinue to dwindle due to antimicrobial resistance development. Thereis a clear demand for new and rapid on-site molecular diagnostic testsenabling identification of specific pathogens.

The Polymerase Chain Reaction (PCR) in many ways revolutionized themolecular genetics and diagnosis field. The workhorses in PCRtechnology, are thermostable high fidelity DNA polymerases which,together with cyclic events of heating and cooling to obtain strandseparation, primer annealing and elongation, lead to amplification of atarget DNA sequence. PCR technology is now widely employed in biomedicaland life science research as well as molecular diagnostics.

Point-of-care (POC) diagnostics are described as medical tools ordevices enabling disease diagnosis in a patient's community outside ahospital setting. The ideal diagnostic test should meet the “ASSURED”criteria: Affordable, Sensitive, Specific, User-friendly, Rapid androbust, Equipment-free and Delivered to those who need it. POC methodsare preferably simple and do not require a heat source or stable powersupply as these are typically not available at POC. Thus enzymes andreagents used should work at ambient temperatures.

Although PCR technology has a high potential, it still has strictlimitations and requires the use of high precision electrically poweredthermal cycling equipment for repeated heating and cooling processes andskilled personnel to run the equipment. Non-specific amplification dueto spurious priming in the annealing process is problematic, and PCR isalso prone to inhibitory compounds in “crude” samples. In addition, thebulky design of PCR devices make PCR an imperfect solution forincorporation into POC technology platforms and make PCR-based methodsdifficult to employ as the major technology driver in POC diagnostics.

Lately, an increased focus on non-PCR based methods, in particularIsothermal Amplification (IA) methods, has emerged. In these methods,nucleic acid amplification takes place at constant temperatures and hasno need for high precision temperature cycling and control, or enzymesstable at high temperatures. Isothermal amplification methods arereported to have analytical sensitivities and specificities comparableto PCR as well as a higher tolerance to inhibitory compounds, whileallowing shorter time to results and easier use. These features makeisothermal amplification methods highly desirable for those developingPOC molecular diagnostics platforms and aiming to meet “ASSURED”criteria. A number of different methods have in the last decade beenpublished for isothermal amplification of nucleic acids (both RNA andDNA) (Reviewed by Gill, P. and A. Ghaemi (2008) Nucleosides NucleotidesNucleic Acids 27(3): 224-243; Craw, P. and W. Balachandran (2012) LabChip 12(14): 2469-2486; de Paz, H. D. et al. (2014) Expert Rev Mol Diagn14(7): 827-843; Yan, L. et al. (2014) Mol Biosyst 10(5): 970-1003 andnew ones are continuously being developed (Liu, W. et al. (2015) SciReports 5: 12723). In several of the methods, success relies on theinherent strand displacement activity (SDA) of the DNA polymerase usedin the reaction setup. The term strand displacement describes theability of the polymerase to displace downstream DNA encountered duringsynthesis.

In addition to (POC) diagnostics also other areas of interest benefitfrom isothermal amplification technology empowered by the DNApolymerase. In this regard, whole genome amplification (multipledisplacement amplification) is important especially when extremelylimited amount of DNA is present such as in single cell approaches.Also, in next-generation sequencing approaches strand-displacingpolymerases are important as exemplified by the Pacific BiosciencesSingle Molecule Real Time (SMRT) DNA sequencing technology and anisothermal amplification method for next generation sequencing publishedin 2013 by Ma et al. (Ma, Z. et al. Proc Natl Acad Sci USA 110(35):14320-14323).

The current toolbox of polymerase enzymes which function well at ambienttemperature is, however, very limited. Typically, different isothermalmethods require reaction temperatures between 30-65° C. which are mainlydetermined by the working range of the polymerases used in the reactionsand are prone to inhibition by salt.

A cold-adapted polymerase from a Psychrobacillus sp. (PB) belonging tothe A-family of DNA polymerases has been characterized. This enzymepossesses high polymerase activity at ambient temperatures but still hasgood stability at elevated temperatures up to 40° C. Of particularinterest, the marine derived enzyme also possesses good salt toleranceand strong strand-displacement activity (SDA) as well as proficientprocessivity at 25° C., and is comparable with the state-of-the artcommercial enzymes (WO 2017/162765).

In many IA methods only a polymerase is required and the effectivenessof the method is heavily dependent on the SDA of that polymerase.Therefore anything which served to increase SDA of the PB or otherpolymerases used in IA would be highly desirable.

The present inventors have surprisingly found that a single pointmutation in the finger domain of certain polymerases in the A family, inparticular replacement of a single Asp residue, leads to significantlyenhanced SDA.

Therefore, in a first aspect, the present invention provides a DNApolymerase, said DNA polymerase including the sequence of SEQ ID NO. 1or a sequence which is at least 70%, preferably at least 75%, 78%, 80%,82%, 85%, 88%, 90%, 92% or 95%, identical thereto, but wherein theaspartic acid residue at position 18 of SEQ ID NO. 1, or the equivalentaspartic acid residue in other sequences, has been replaced by anon-negatively charged amino acid residue.

SEQ ID NO. 1 is a fragment of the amino acid sequence of the PBpolymerase I, it spans amino acids 405 to 436 in the truncated (lackingthe 5′-3′-exonuclease domain) wild type PB sequence. This region(405-436) within the finger domain is highly conserved amongst some ofthe DNA polymerase A family (also known as pol I family), see FIGS. 1and 5 . DNA polymerases of the invention would typically be classed asof the DNA polymerase I or A type.

Preferred DNA polymerases of the invention comprise the sequence of SEQID NO. 6, 7, 8, 9 or 10 but wherein the aspartic acid residue atposition 18 of each sequence has been replaced by a non-negativelycharged amino acid residue.

In some embodiments, the DNA polymerase of the invention comprises anamino acid sequence that has single or multiple amino acid alterations(additions, substitutions, insertions or deletions) compared to SEQ IDNO:1. Such sequences preferably may contain up to 8, 7 or 6, e.g. only1, 2, 3, 4 or 5, preferably 1, 2 or 3, more preferably 1 or 2, alteredamino acids in addition to the replacement of the aforementioned Aspresidue. Substitutions can be with conservative or non-conservativeamino acids. Preferably said alterations are conservative amino acidsubstitutions.

A preferred polymerase of the invention is a modified PB polymerase,further preferred polymerases are modified polymerases from the speciesGeobacillus stearothermophilus (known as Bst), from Bacillus subtilis(known as Bsu), from Bacillus smithii (known as Bsm) and Ureibacillusthermosphaericus (known as Ubts).

The term “DNA polymerase” refers to an enzyme which catalyses the 5′→3′synthesis of DNA from individual nucleotides, the reaction being basedon primer extension and standard Watson-Crick rules of base pairing to atemplate strand. Likewise, “DNA polymerase activity” refers to the 5′→3′synthesis of DNA from individual nucleotides, the reaction being basedon primer extension and standard Watson-Crick rules of base pairing to atemplate strand. Enzymatically active (catalytically active) fragmentsof naturally occurring or modified polymerases are included within theterm “DNA polymerase”. The polymerase may also, but may not, have 3′→5′exonuclease and/or 5′→3′ exonuclease activity. Preferably the DNApolymerases of the present invention lack 5′→3′ exonuclease activity.

The present inventors have found that replacement of the aforementionedaspartic acid residue significantly increases SDA in several differentDNA polymerases in the family known as DNA polymerase A, the enzymeswhich may benefit from modification in accordance with the presentinvention are characterised by a high sequence identity with SEQ ID NO.1 (a particular region of the finger domain of the PB enzyme) and anaspartic acid residue at position 18 or the equivalent position in otherenzymes/sequences. An “equivalent aspartic acid residue in othersequences” than SEQ ID NO. 1 (or other sequences) can be readilyidentified by using standard sequence alignment techniques such asClustal X2 (Larkin, M. A. et al. (2007) Clustal W and Clustal X version2.0. Bioinformatics, 23:2947-2948).

Of course, SEQ ID NO. 1 does not itself define a fully functional DNApolymerase. In preferred embodiments the DNA polymerase of the presentinvention is based on the amino acid sequence of PB DNA polymerase I,preferably which lacks the 5′-3′-exonuclease domain that is present inthe wild-type Psychrobacillus species DNA polymerase I sequence. Inpreferred embodiments, the 5′-3′-exonuclease domain is absent from theDNA polymerase enzyme as 5′-3′-exonuclease activity is typicallyunwanted as it may degrade primers and/or products in an amplificationmixture. This truncated wild-type PB sequence is referred to herein asSEQ ID NO. 2.

The invention provides a DNA polymerase comprising or consisting of theamino acid sequence of SEQ ID NO:2 or an amino acid sequence which is atleast 60% identical to SEQ ID NO:2 but wherein the aspartic acid residueat position 422 of SEQ ID NO. 2, or the equivalent aspartic acid residuein other sequences, has been replaced by a non-negatively charged aminoacid residue.

In preferred aspects and embodiments, the DNA polymerase of theinvention comprises (or consists of) an amino acid sequence that is atleast 70%, or 75%, preferably at least 80%, 85%, 90% or 95%, e.g. atleast 98% or 99% or 99.5%, identical to SEQ ID NO:2 but wherein theaspartic acid residue at position 422 of SEQ ID NO. 2, or the equivalentaspartic acid residue in other sequences, has been replaced by anon-negatively charged amino acid residue. It will be understood thatposition 18 in SEQ ID NO. 1 and 422 in SEQ ID NO. 2 are equivalent, SEQID NO. 1 is a fragment from position 405 to 436 of SEQ ID NO. 2.

FIG. 5 shows an alignment of PB, Bst and Ubts DNA polymerases. The keyAsp residue is at position 422 in each case.

In further preferred embodiments or aspects, the DNA polymerase of theinvention comprises (or consists of) an amino acid sequence that is atleast 60%, 70% or 75%, preferably at least 80%, 85%, 90% or 95%, e.g. atleast 98% or 99% or 99.5%, identical to SEQ ID NO. 11 or 12 but whereinthe aspartic acid residue at position 422 of SEQ ID NO. 11 or 12, or theequivalent aspartic acid residue in other sequences, has been replacedby a non-negatively charged amino acid residue. Numbering is based on asequence alignment according to FIG. 5 . SEQ ID NOs. 11 and 12 are the(truncated) variants of the wild type Bst and Ubts polymerase sequencesrespectively.

Preferably, the DNA polymerase of the invention comprises or consists ofthe amino acid sequence of SEQ ID NO:2, 11 or 12 but wherein theaspartic acid residue at position 422 of SEQ ID NO. 2, 11 or 12, or theequivalent aspartic acid residue in other sequences, has been replacedby a non-negatively charged amino acid residue.

In one embodiment, the DNA polymerase comprises (or consists of) theamino acid sequence of SEQ ID NO:4 (incorporating also the exonucleasedomain) or a variant or fragment thereof but wherein the aspartic acidresidue at position 719 of SEQ ID NO. 4, or the equivalent aspartic acidresidue in other sequences, has been replaced by a non-negativelycharged amino acid residue. The types of variants and fragments of SEQID NO:2 described herein apply, mutatis mutandis, to variants andfragments of SEQ ID NO:4, e.g. variants will have at least 70%preferably at least 80% or 90% sequence identity to SEQ ID. NO:4.

DNA polymerases of the invention include enzymatically active fragmentsof native polymerases. Enzymatically active fragments are fragments thathave DNA polymerase activity. Enzymatically active fragments may be atleast 400, at least 450, at least 475, at least 500, at least 525, atleast 550, at least 560, at least 570 or at least 575 amino acids inlength. Preferred fragments are at least 525, at least 550, at least560, at least 570 or at least 575 amino acids in length. The fragmentsare at least 70%, preferably at least 80%, at least 85% or at least 90%,more preferably at least 95% (e.g. at least 98% or 99% or 99.5%), or100% identical to the corresponding portion of SEQ ID NO:2, 11 or 12 butwherein the aspartic acid residue at position 422 of SEQ ID NO. 2, 11 or12, or the equivalent aspartic acid residue in other sequences, has beenreplaced by a non-negatively charged amino acid residue.

DNA polymerase activity may be assessed using a molecular beacon thatbears a loop structure and uses FAM as fluorescence donor and Dabcyl asan acceptor (non-fluorescent quencher) within an 8mer stem. This stembears a 3′-extension that allows binding of a primer and acts astemplate for the DNA polymerase. The stem will be opened by the DNApolymerase when the extension proceeds. The following separation of thetwo labels is recorded by restoration of FAM emission. A suitable assayof this type is described in the Examples.

The DNA polymerases of the present invention have good stranddisplacement activity. This is an important property as in manyisothermal amplification methods success relies on the inherent stranddisplacement activity of the DNA polymerase used in the reaction setup.The term “strand displacement” describes the ability of the polymeraseto displace downstream DNA encountered during synthesis.

Suitable assays to assess strand displacement activity of a DNApolymerase are known in the art and a skilled person is readily able toselect a suitable assay. In an exemplary strand displacement activityassay, a “cold” primer and a reporter strand that is labelled with afluorophore (e.g. TAM RA) at its 3′ end are annealed to a templatestrand that has a quencher (e.g. BHQ2) at its 5′ end (the fluorophore isthus quenched by the close proximity of the quencher) such that there isa one nucleotide gap between the 3′ end of the annealed “cold” primerand the 5′ end of the annealed reporter strand; upon strand displacementactivity of the DNA polymerase the fluorophore labelled oligonucleotide(reporter strand) is displaced from the template strand and as aconsequence the fluorophore and quencher are no longer in closeproximity and an increase in fluorescence can be measured.

Strand displacement activity may be assessed in an assay having thesteps of (i) providing a template DNA molecule that has a quencher(fluorescence quencher) at its 5′ end, (ii) annealing to said templateDNA molecule a cold primer (i.e. non-fluorescent oligonucleotide) and areporter strand (reporter oligonucleotide) that is labelled with afluorophore at its 3′ end wherein there is a one nucleotide gap betweenthe 3′ end of the annealed “cold” primer and the 5′ end of the annealedreporter strand, whereby the quencher quenches the fluorophore by virtueof their close proximity to each other, (iii) incubating saidtemplate-cold primer-reporter strand complex with a DNA polymerase, Me²⁺and dNTPs and (iv) measuring the increasing fluorescence of thepreviously quenched fluorophore, wherein said fluorescence is indicativeof strand displacement activity.

Preferred primers, reporter strands and template strands are asdescribed in the Examples.

In a preferred embodiment strand displacement activity (SDA) is asassessed in accordance with the strand displacement activity assaydescribed in the Example section. SDA is preferably measured at aboutthe optimum temperature for that polymerase. For PB and other mesophilesthat may be around 25° C.-37° C.

The present invention allows the SDA of a wild type DNA polymerase to beenhanced. In the case of PB the SDA is already high compared to mostcommercially available polymerases but SDA can still be significantlyincreased (see FIG. 3 ) by implementing the amino acid modification atposition 18/422 described herein. For thermostable polymerases includedin the invention, e.g. Ubts and Bst, the native SDA is quite low atambient temperatures (25-37° C.). However the SDA is still significantlyenhanced at 37° C. when the aspartic acid residue is replaced with anon-negatively charged amino acid residue. Different polymerases may beuseful in different scenarios, e.g. Bst and Ubts are thermostable (Tm of66° C. and 62° C. respectively), and so the ability to enhance SDA forany of these enzymes is very useful (see FIGS. 6 and 7 ).

Thus, in preferred embodiments, the DNA polymerases of the inventionhave at least 30%, preferably at least 50%, more preferably at least100% greater SDA than a DNA polymerase with exactly the same sequencebut with aspartic acid at position 18 or 422, relative to SEQ ID NOs. 1and 2 respectively, or at the equivalent position in other sequences.Preferably the % increase observed will at least be seen under theconditions at which each enzyme exhibits its maximum SDA. In otherwords, for the best that each enzyme can perform, the polymerase of theinvention will preferably have at least 30%, more preferably at least50%, most preferably at least 100% higher SDA than its aspartic acidcontaining equivalent.

The aspartic acid residue discussed above, the modification of which iskey to the benefits provided by the present invention, is replaced by aresidue without a negative charge. The replacement will typicallyinvolve substitution with another amino acid residue but in someembodiments the aspartic acid residue may have been modified to removeits negative charge. Thus, the residue at position 18/422 of thepolymerase of the invention will be either neutral or positivelycharged. Neutral amino acids include polar amino acids and hydrophobicamino acids. Suitable replacement amino acids include Ser, Thr, Asn,Gln, Ala, Ile, Leu, Tyr, Val, Lys and Arg. Non-standard, i.e.non-genetically coded amino acids, may be incorporated which are neutralor positively charged. Ala is particularly preferred.

The inventors have also found that some of the polymerases of theinvention exhibit improved SDA performance at elevated [NaCl] and [KCl]as compared to enzymes which contain the Asp residue discussed above(see tables 2 and 4). Enhanced salt tolerance is thus a further benefitwhich may be provided by the present invention.

Preferred DNA polymerases of the present invention have useful levels ofpolymerase activity across a range of salt (NaCl and/or KCl)concentrations. Put another way, preferred DNA polymerases of thepresent invention exhibit across a broad range of salt concentrations asubstantial proportion of the DNA polymerase activity observed at thesalt concentration at which maximum polymerase activity is observed.Suitable assays for determining DNA polymerase activity are describedelsewhere herein. A preferred assay for determining DNA polymeraseactivity is as described in the Example section.

In some embodiments, across a concentration range from about 20 mM to200 mM NaCl or KCl or a mixture thereof, DNA polymerases of the presentinvention exhibit a substantial proportion (e.g. at least 40%,preferably at least 50%, more preferably at least 60%) of their maximumpolymerase activity.

In a further aspect the present invention provides molecules (e.g.proteins, such as fusion proteins) comprising DNA polymerases of thepresent invention.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit or exclusion is thereafterspecifically stated. The operable limits and parameters of combinations,as with the amounts of any single agent, will be known to those ofordinary skill in the art in light of the present disclosure.

Nucleic acid molecules comprising nucleotide sequences that encode DNApolymerases of the present invention as defined herein or fragmentsthereof, or nucleic acid molecules substantially homologous thereto,form yet further aspects of the invention. A preferred nucleic acidmolecule is a nucleic acid encoding a DNA polymerase I of SEQ ID NO:2,or a sequence substantially homologous thereto (e.g. at least 60%, 70%,75%, 80%, 85%, 90% or 95% identical thereto), but wherein the asparticacid residue at position 422 of SEQ ID NO. 2, or the equivalent asparticacid residue in other sequences, has been replaced by a non-negativelycharged amino acid residue.

A preferred nucleic acid molecule comprises (or consists of) thenucleotide sequence as set forth in SEQ ID NO: 13, 14 or 15, or is asequence substantially homologous thereto. Optionally, the final threenucleotides of SEQ ID NO: 13, 14 or 15 may be omitted. Nucleic acidsequences of the invention include sequences having at least 70% or 75%,preferably at least 80%, and even more preferably at least 85%, 90%,95%, 96%, 97%, 98%, 99% or 99.5%, sequence identity to SEQ ID NO: 13, 14or 15. Nucleic acid sequences of the invention thus include single ormultiple base alterations (additions, substitutions, insertions ordeletions) to the sequence of SEQ ID NO: 13, 14 or 15.

A particularly preferred nucleic acid molecule comprises or consists ofthe nucleotide sequence as set forth in SEQ ID NO: 13, 14 or 15.

The present invention also extends to nucleic acid molecules comprising(or consisting of) nucleotide sequences which are degenerate versions ofnucleic acid molecules described herein, e.g. degenerate versions of anucleic acid molecule comprising (or consisting of) SEQ ID NO: 13, 14 or15.

Nucleic acid molecules of the invention are preferably “isolated” or“purified”.

Homology (e.g. sequence identity) may be assessed by any convenientmethod. However, for determining the degree of homology (e.g. identity)between sequences, computer programs that make multiple alignments ofsequences are useful, for instance Clustal W (Thompson, Higgins, Gibson,Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal Walgorithm can be used together with BLOSUM 62 scoring matrix (Henikoffand Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and agap opening penalty of 10 and gap extension penalty of 0.1, so that thehighest order match is obtained between two sequences wherein at least50% of the total length of one of the sequences is involved in thealignment. Clustal X is a convenient windows interface for Clustal W(Thompson, J. D. et al (1997) The ClustalX windows interface: flexiblestrategies for multiple sequence alignment aided by quality analysistools. Nucleic Acids Research, 25:4876-4882).

Other methods that may be used to align sequences are the alignmentmethod of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol.,48:443, 1970) as revised by Smith and Waterman (Smith and Waterman, Adv.Appl. Math., 2:482, 1981) so that the highest order match is obtainedbetween the two sequences and the number of identical amino acids isdetermined between the two sequences. Other methods to calculate thepercentage identity between two amino acid sequences are generally artrecognized and include, for example, those described by Carillo andLipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988) andthose described in Computational Molecular Biology, Lesk, e.d. OxfordUniversity Press, New York, 1988, Biocomputing: Informatics and GenomicsProjects.

Generally, computer programs will be employed for such calculations.Programs that compare and align pairs of sequences, like ALIGN (Myersand Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc.Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods inEnzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., NucleicAcids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux,Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also usefulfor this purpose. Furthermore, the Dali server at the EuropeanBioinformatics institute offers structure-based alignments of proteinsequences (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm,J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9,1998).

By way of providing a reference point, sequences according to thepresent invention having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99%, sequence identity etc. may be determined using the ALIGNprogram with default parameters (for instance available on Internet atthe GENESTREAM network server, IGH, Montpellier, France).

A “conservative amino acid substitution”, as used herein, is one inwhich the amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art.

DNA polymerases of the present invention comprise genetically encodedamino acids, but may also contain one or more non-genetically encodedamino acids.

When used in connection with a protein or polypeptide molecule such as aDNA polymerase, the term “isolated” or “purified” typically refers to aprotein substantially free of cellular material or other proteins fromthe source from which it is derived. In some embodiments, such isolatedor purified proteins are substantially free of culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

In one further aspect the present invention provides an expressionvector (preferably a recombinant expression vector) containing a nucleicacid molecule of the invention, or a fragment thereof, and the necessaryregulatory sequences for the transcription and translation of theprotein sequence encoded by the nucleic acid molecule of the invention.

Possible expression vectors include but are not limited to cosmids orplasmids, so long as the vector is compatible with the host cell used.The expression vectors are “suitable for transformation of a host cell”,which means that the expression vectors contain a nucleic acid moleculeof the invention and regulatory sequences selected on the basis of thehost cells to be used for expression, which are operatively linked tothe nucleic acid molecule. Operatively linked is intended to mean thatthe nucleic acid is linked to regulatory sequences in a manner thatallows expression of the nucleic acid.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes and arewell known in the art. Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.

The recombinant expression vectors may also contain genes that encode afusion moiety that provides increased expression of the recombinantprotein; increased solubility of the recombinant protein and/or aids inthe purification of the target recombinant protein by acting as a ligandin affinity purification (for example appropriate “tags” to enablepurification and/or identification may be present, e.g., His tags or myctags).

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. The terms “transformed with”,“transfected with”, “transformation” and “transfection” are intended toencompass introduction of nucleic acid (e.g., a vector) into a cell byone of many possible techniques known in the art. Suitable methods fortransforming and transfecting host cells can be found in Sambrook etal., 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989) and other laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic host cells andeukaryotic cells. Preferably, proteins of the invention may be expressedin bacterial host cells, such as Escherichia coli.

N-terminal or C-terminal fusion proteins comprising DNA polymerases andproteins of the invention conjugated to other molecules, such asproteins (e.g. epitope tags), may be prepared by fusing throughrecombinant techniques.

A yet further aspect provides a host cell or virus comprising one ormore expression constructs or expression vectors of the invention. Alsoprovided are host cells or viruses comprising one or more of the nucleicacid molecules of the invention. A host cell or virus capable ofexpressing a DNA polymerase of the invention forms a yet further aspect.Preferred host cells include Rosetta 2 (DE3) cells (Novagen).

DNA polymerases of the invention may be produced recombinantly in a hostcell and isolated and purified therefrom. The DNA polymerases of theinvention may therefore be considered recombinant enzymes, in particularisolated recombinant enzymes. In certain embodiments the DNA polymeraseis produced by recombinant techniques in a host cell that is not, or notfrom, an organism which is the same as that from which the DNApolymerase was derived.

DNA polymerases of the present invention may be generated usingrecombinant DNA technology. Alternatively, a cell-free expression systemcan be used for production of the DNA polymerase. Alternatively, DNApolymerases of the present invention may be generated using chemicalsynthesis so that the DNA polymerase is generated by stepwiseelongation, one amino acid at a time. Such chemical synthesis techniques(e.g. solid phase synthesis) are well known in the chemistry ofproteins.

A further aspect of the invention provides a method of producing a DNApolymerase of the present invention comprising a step of culturing thehost cells of the invention. Preferred methods comprise the steps of (i)culturing a host cell comprising one or more of the recombinantexpression vectors or one or more of the nucleic acid molecules of theinvention under conditions suitable for the expression of the encodedDNA polymerase or protein; and optionally (ii) isolating or obtainingthe DNA polymerase or protein from the host cell or from the growthmedium/supernatant. Such methods of production may also comprise a stepof purification of the DNA polymerase or protein product and/orformulating the DNA polymerase or product into a composition includingat least one additional component, such as an acceptable buffer orcarrier.

The DNA polymerase may be separated, or isolated, from the hostcells/culture media using any of the purification techniques for proteinknown in the art and widely described in the literature or anycombination thereof. Such techniques may include for example,precipitation, ultrafiltration, dialysis, various chromatographictechniques, e.g. size exclusion chromatography, ion-exchangechromatography, affinity chromatography, electrophoresis, centrifugationetc. As discussed above, the DNA polymerase of the invention may bemodified to carry amino acid motifs or other protein or non-proteintags, e.g. polyhistidine tags (e.g. His₆-tag), to assist in isolation,solubilisation and/or purification or identification.

In another aspect, the present invention provides the use of a DNApolymerase of the invention for nucleotide (e.g. dNTP) polymerisation.Accordingly, DNA polymerases of the invention may be used to extend anucleic acid (DNA) strand by one or more nucleotides.

In another aspect, the present invention provides the use of a DNApolymerase of the invention in a nucleic acid (DNA) amplification orsequencing reaction.

In another aspect, the present invention provides the use of a DNApolymerase of the invention in a molecular beacon assay or in a stranddisplacement assay, e.g. as described herein.

Preferably, in uses and methods of the present invention, DNApolymerases of the present invention are used at a constant temperature,i.e. without thermal cycling. Accordingly, the use of DNA polymerases ofthe invention in isothermal reactions is particularly preferred.

The use of DNA polymerases of the invention in isothermal amplificationreactions is particularly preferred. Isothermal reactions are performedat a constant temperature. Many isothermal amplification techniques areknown in the art and include Loop mediated isothermal amplification(LAMP), rolling circle amplification (RCA), strand displacementamplification (SDA), multiple displacement amplification (MDA) and crosspriming amplification (CPA).

In another aspect, the present invention provides a method of nucleotidepolymerisation using a DNA polymerase of the present invention.Preferably, said method comprises providing a reaction mixturecomprising a DNA polymerase of the present invention, a template nucleicacid molecule, an oligonucleotide primer which is capable of annealingto a portion of the template nucleic acid molecule and one or morespecies of nucleotide (e.g. deoxynucleoside triphosphates, dNTPs) andincubating said reaction mixture under conditions whereby theoligonucleotide primer anneals to the template nucleic acid molecule andsaid DNA polymerase extends said oligonucleotide primer by polymerisingone or more nucleotides. Suitable conditions are well known in the art.Preferably a constant temperature is used and preferred temperatures areset out elsewhere herein. Optionally, the generation of thepolynucleotide product is detected (e.g. via gel electrophoresis).

In another aspect, the present invention provides a method of amplifyinga nucleic acid (DNA) using a DNA polymerase of the present invention.Typically, said method comprises providing a reaction mixture comprisinga DNA polymerase of the present invention, a template nucleic acidmolecule, an oligonucleotide primer(s) (e.g. 2 or more primers such as2, 3, 4, 5 or 6 primers) which is capable of annealing to a portion ofthe template nucleic acid molecule acid molecule, and nucleotides (e.g.deoxynucleoside triphosphates, dNTPs) and incubating said reactionmixture under conditions whereby the oligonucleotide primer(s) annealsto the template nucleic acid molecule and said DNA polymerase extendssaid oligonucleotide primer(s) by polymerising one or more nucleotidesto generate a polynucleotide. Suitable conditions are well known in theart. Preferred methods of nucleic acid amplification are isothermalamplification methods. Isothermal amplification methods of the inventionare performed at a constant temperature and preferred temperatures areset out elsewhere herein. Optionally, the generation of thepolynucleotide product is detected (e.g. via gel electrophoresis).

Exemplary isothermal amplification methods include Loop mediatedisothermal amplification (LAMP), rolling circle amplification (RCA),strand displacement amplification (SDA), multiple displacementamplification (MDA) and cross priming amplification (CPA).

In some embodiments, particularly those using DNA polymerases based onthe PB sequence, the constant temperature used in the methods and usesof the present invention is a low-to-moderate temperature, for example,is chosen from within the range 0° C. to about 42° C., preferably ischosen from within the range about 10° C. to about 40° C., or about 20°C. to about 40° C., or about 25° C. to about 40° C., or about 30° C. toabout 40° C. or about 35° C. to about 40° C., or about 37° C. to about40° C. In some embodiments, the constant temperature is chosen fromwithin the range about 10° C. to about 15° C., or about 10° C. to about20° C. In some embodiments, the constant temperature is chosen fromwithin the range about 10° C. to about 30° C. In some embodiments, theconstant temperature is chosen from within the range about 20° C. toabout 30° C. In some embodiments, the constant temperature is chosenfrom within the range about 10° C. to about 25° C. In some embodiments,the constant temperature is chosen from within the range about 20° C. toabout 25° C. A constant temperature of about 25° C. is preferred. Insome embodiments, the constant temperature is 25° C.

With other polymerases of the invention, for example those based onsequences from organisms which are thermophilic, the constanttemperature may be moderate to high, e.g. is chosen from within therange 25° C.-65° C., preferably 40° C.-65° C.

A temperature may be considered constant when no active steps are takento modify the temperature during the reaction, e.g. no thermal cycling.A ‘constant’ temperature may still allow temperature fluctuations duringthe method e.g. of up to about 5° C., typically no more than 3° C. or 2°C.

DNA polymerases of the present invention may be used in point-of-caremolecular diagnostics platforms.

DNA polymerases of the present invention may be used in whole genomeamplification.

DNA polymerases of the present invention may be used in next-generationsequencing methods. So-called “next generation” or “second generation”sequencing approaches (in reference to the Sanger dideoxynucleotidemethod as the “first generation” approach) have become widespread. Thesenewer techniques are characterised by high throughputs, e.g. as aconsequence of the use of parallel, e.g. massively parallel sequencingreactions, or through less time-consuming steps. Various high throughputsequencing methods provide single molecule sequencing and employtechniques such as pyrosequencing, reversible terminator sequencing,cleavable probe sequencing by ligation, non-cleavable probe sequencingby ligation, DNA nanoballs, and real-time single molecule sequencing.

References herein to DNA polymerases of the invention encompass activefragments unless otherwise clear from the context.

Uses and methods of the present invention are typically performed invitro.

The present invention also provides compositions comprising a DNApolymerase of the invention. Such compositions preferably comprise abuffer. Optionally, compositions of the present invention furthercomprise one or more of the necessary reagents to carry out a nucleicacid amplification reaction (e.g. an isothermal amplification reaction),e.g. oligonucleotide primers capable of annealing to a region of thetemplate DNA to be amplified and/or nucleotides (e.g. dNTPs). Typicallycompositions will be aqueous and buffered with a standard buffer such asTris, HEPES, etc.

The invention further includes kits comprising one or more of the DNApolymerases of the invention, or one or more compositions of theinvention, or one or more of the nucleic acid molecules of theinvention, or one or more expression vectors of the invention, or one ormore host cells or viruses of the invention. Preferably said kits arefor use in the methods and uses as described herein, e.g., in nucleicacid amplification methods, such as isothermal amplification reactions.Preferably said kits comprise instructions for use of the kitcomponents, for example for nucleic acid amplification.

Nucleotide and Amino Acid Sequences Disclosed Herein and their SequenceIdentifiers (SEQ ID NOs)

All nucleotide sequences are recited herein 5′ to 3′ in line withconvention in this technical field.

SEQ ID NO: 1-amino acid sequence of a region of the fingerdomain of DNA polymerase I from a Psychrobacillus sp. SEQ ID NO: 1MRRAAKAVNFGIVYGISDYGLSQNLDITRKEASEQ ID NO: 2-amino acid sequence of truncated DNApolymerase I isolated from a Psychrobacillus sp. (PB) SEQ ID NO: 2TEVAFEIVEEIDSTILDKVMSVHLEMYDGQYHTSELLGIALSDGEKGYFAPADIAFQSKDFCSWLENATNKKYLADSKATQAVSRKHNVNVHGVEFDLLLAAYIVNPAISSEDVAAIAKEFGYFNLLTNDSVYGKGAKKTAPEIEKIAEHAVRKARAIWDLKEKLEVKLEENEQYALYKEIELPLASILGTMESDGVLVDKQILVEMGHELNIKLRAIEQDIYALAGETFNINSPKQLGVILFEKIGLTPIKKTKTGYSTAADVLEKLASEHEIIEQILLYRQLGKLNSTYIEGLLKEIHEDDGKIHTRYQQALTSTGRLSSINPNLQNIPVRLEEGRKIRKAFVPSQPGWVMFAADYSQIELRVLAHMSEDENLVEAFNNDLDIHTKTAMDVFHVEQEAVTSDMRRAAKAVNFGIVYGISDYGLSQNLDITRKEAATFIENYLNSFPGVKGYMDDIVQDAKQTGYVTTILNRRRYLPEITSSNFNLRSFAERTAMNTPIQGSAADIIKKAMIDMAERLISENMQTKMLLQVHDELIFEAPPEEIAMLEKIVPEVMENAIKLIVPLKVDYAFG SSWYDTKSEQ ID NO: 3-nucleic acid sequence encoding thePsychrobacillus species DNA polymerase I sequence of SEQ ID NO: 2SEQ ID NO: 3 ACAGAAGTAGCATTCGAGATTGTTGAAGAAATTGACTCTACAATATTAGATAAAGTAATGTCAGTCCATTTAGAAATGTATGATGGGCAATATCATACAAGCGAATTATTAGGTATTGCTTTATCAGATGGAGAAAAGGGTTATTTTGCTCCTGCTGATATAGCTTTTCAATCGAAGGATTTTTGTTCTTGGTTAGAAAATGCTACGAATAAAAAGTATTTAGCAGACTCCAAAGCAACACAAGCAGTGAGTAGAAAACATAATGTGAATGTACATGGAGTGGAATTCGACCTTCTTTTAGCAGCGTATATAGTAAATCCTGCTATCTCTTCAGAGGATGTTGCTGCTATTGCTAAAGAATTTGGATATTTTAACTTGCTGACAAACGATAGTGTTTATGGGAAAGGTGCCAAAAAAACCGCACCTGAAATCGAGAAAATTGCAGAACATGCCGTAAGAAAAGCAAGGGCTATTTGGGACTTGAAAGAAAAGTTAGAAGTAAAACTGGAAGAAAATGAACAATATGCGTTGTATAAAGAAATAGAGCTACCGCTTGCATCTATCCTTGGTACGATGGAATCAGATGGGGTGCTGGTGGATAAACAAATTCTTGTAGAAATGGGTCATGAGCTTAATATTAAGTTACGAGCGATTGAACAAGACATTTATGCGTTAGCTGGTGAAACGTTTAATATTAATTCACCTAAACAATTAGGTGTAATACTATTTGAAAAAATTGGTCTTACCCCTATTAAAAAGACAAAAACGGGCTATTCAACTGCAGCAGATGTTTTGGAAAAACTAGCAAGTGAACATGAAATAATAGAGCAAATTTTACTATATCGTCAATTAGGTAAACTCAATTCCACATATATCGAAGGATTATTAAAAGAGATTCATGAAGATGATGGGAAGATCCATACCCGATATCAACAAGCCCTAACTTCAACTGGGCGTTTGAGTTCGATCAATCCAAACCTTCAAAATATACCAGTTCGTTTAGAAGAAGGTAGAAAAATACGTAAAGCCTTTGTTCCTTCACAACCGGGATGGGTAATGTTTGCGGCGGATTACTCTCAAATTGAATTGCGTGTTCTTGCCCATATGTCTGAGGATGAAAACCTGGTAGAAGCTTTTAATAATGATCTGGATATTCATACTAAAACGGCTATGGATGTATTCCATGTGGAGCAGGAAGCAGTAACGTCCGATATGCGCCGTGCTGCTAAGGCAGTTAACTTTGGGATTGTGTATGGTATTAGTGATTATGGTTTATCACAAAACCTAGATATTACTAGAAAAGAAGCGGCGACATTTATCGAGAATTATTTAAATAGCTTCCCAGGTGTAAAAGGATATATGGATGATATCGTTCAAGATGCGAAACAAACAGGCTACGTTACAACAATTTTGAATAGACGAAGATATTTGCCTGAAATAACAAGTTCTAACTTTAATCTCCGCAGTTTTGCAGAACGTACTGCTATGAATACACCAATTCAAGGGAGTGCAGCCGATATTATTAAAAAAGCAATGATCGATATGGCGGAAAGATTAATATCAGAAAATATGCAGACCAAAATGCTACTACAAGTACATGATGAATTAATTTTTGAGGCTCCACCAGAGGAAATTGCAATGCTAGAAAAAATAGTGCCAGAGGTGATGGAAAACGCTATTAAACTGATTGTACCTTTGAAAGTGGATTATGCCTTTGGTTCATCTTGGTATGACACGAAGTAGSEQ ID NO: 4-amino acid sequence of full-length DNApolymerase I isolated from a Psychrobacillus sp. SEQ ID NO: 4MYLSTEKILLLDGNSLAYRAFFALPLLTNEHGIHTNAVYGFTMMLQKIMDEENPTHMLVAFDAGKTTFRHSTFGDYKGGRQKTPPELSEQFPYIRKLIDAYGIKRYELEMYEADDIIGTLSKRADEKGQQVVIVSGDKDLTQLATDKTTVYITRKGITDIEKYTPEHVQEKYGLTPLQIIDMKGLMGDASDNIPGVPGVGEKTAIKLLKEHGSVEDLYKALDTVSGVKLKEKLIANEEQAIMSKALATIETAAPIQISIDDLSYTGPNMEEVIEVWKELAFKTLLEKSDYISEESETTEVAFEIVEEIDSTILDKVMSVHLEMYDGQYHTSELLGIALSDGEKGYFAPADIAFQSKDFCSWLENATNKKYLADSKATQAVSRKHNVNVHGVEFDLLLAAYIVNPAISSEDVAAIAKEFGYFNLLTNDSVYGKGAKKTAPEIEKIAEHAVRKARAIWDLKEKLEVKLEENEQYALYKEIELPLASILGTMESDGVLVDKQILVEMGHELNIKLRAIEQDIYALAGETFNINSPKQLGVILFEKIGLTPIKKTKTGYSTAADVLEKLASEHEIIEQILLYRQLGKLNSTYIEGLLKEIHEDDGKIHTRYQQALTSTGRLSSINPNLQNIPVRLEEGRKIRKAFVPSQPGWVMFAADYSQIELRVLAHMSEDENLVEAFNNDLDIHTKTAMDVFHVEQEAVTSDMRRAAKAVNFGIVYGISDYGLSQNLDITRKEAATFIENYLNSFPGVKGYMDDIVQDAKQTGYVTTILNRRRYLPEITSSNFNLRSFAERTAMNTPIQGSAADIIKKAMIDMAERLISENMQTKMLLQVHDELIFEAPPEEIAMLEKIVPEVMENAIKLIVPLKVDYAFGSSWYDTKSEQ ID NO: 5-nucleic acid sequence encoding thePsychrobacillus sp. DNA polymerase 1 sequence of SEQ ID NO: 4.SEQ ID NO: 5 ATGTATTTGTCAACCGAGAAAATCCTATTATTAGACGGCAATAGTTTGGCATACCGAGCTTTTTTTGCCCTACCTTTATTAACAAATGAACATGGAATACATACAAACGCAGTATATGGCTTTACAATGATGCTACAAAAAATTATGGATGAAGAAAATCCTACTCATATGCTCGTGGCATTTGATGCCGGGAAAACGACCTTCCGTCACTCTACTTTTGGGGATTATAAAGGTGGAAGACAAAAAACACCACCAGAACTATCGGAACAATTCCCTTATATACGCAAGTTAATCGATGCTTATGGTATTAAGCGATACGAACTGGAAATGTACGAAGCAGACGATATTATCGGTACTTTAAGCAAGCGTGCAGACGAAAAAGGGCAGCAAGTTGTAATTGTCTCAGGTGATAAAGATTTAACACAACTAGCTACAGATAAAACAACTGTGTATATCACAAGAAAAGGCATAACCGATATTGAAAAATATACACCTGAACATGTACAAGAAAAGTATGGCTTAACTCCATTACAGATTATAGACATGAAAGGTTTAATGGGAGATGCTTCTGATAATATTCCAGGAGTTCCTGGTGTCGGAGAAAAAACAGCTATTAAGCTTTTAAAAGAACATGGTTCGGTAGAGGATTTATATAAAGCACTTGATACAGTTAGTGGTGTTAAACTAAAGGAAAAACTCATCGCCAACGAAGAGCAGGCAATTATGAGTAAGGCATTAGCTACGATTGAAACAGCTGCACCGATACAGATTTCTATAGACGATCTTTCATATACTGGTCCTAATATGGAAGAAGTAATTGAAGTTTGGAAGGAACTAGCTTTTAAAACTCTTCTTGAGAAATCTGACTATATTTCTGAGGAATCCGAAACTACAGAAGTAGCATTCGAGATTGTTGAAGAAATTGACTCTACAATATTAGATAAAGTAATGTCAGTCCATTTAGAAATGTATGATGGGCAATATCATACAAGCGAATTATTAGGTATTGCTTTATCAGATGGAGAAAAGGGTTATTTTGCTCCTGCTGATATAGCTTTTCAATCGAAGGATTTTTGTTCTTGGTTAGAAAATGCTACGAATAAAAAGTATTTAGCAGACTCCAAAGCAACACAAGCAGTGAGTAGAAAACATAATGTGAATGTACATGGAGTGGAATTCGACCTTCTTTTAGCAGCGTATATAGTAAATCCTGCTATCTCTTCAGAGGATGTTGCTGCTATTGCTAAAGAATTTGGATATTTTAACTTGCTGACAAACGATAGTGTTTATGGGAAAGGTGCCAAAAAAACCGCACCTGAAATCGAGAAAATTGCAGAACATGCCGTAAGAAAAGCAAGGGCTATTTGGGACTTGAAAGAAAAGTTAGAAGTAAAACTGGAAGAAAATGAACAATATGCGTTGTATAAAGAAATAGAGCTACCGCTTGCATCTATCCTTGGTACGATGGAATCAGATGGGGTGCTGGTGGATAAACAAATTCTTGTAGAAATGGGTCATGAGCTTAATATTAAGTTACGAGCGATTGAACAAGACATTTATGCGTTAGCTGGTGAAACGTTTAATATTAATTCACCTAAACAATTAGGTGTAATACTATTTGAAAAAATTGGTCTTACCCCTATTAAAAAGACAAAAACGGGCTATTCAACTGCAGCAGATGTTTTGGAAAAACTAGCAAGTGAACATGAAATAATAGAGCAAATTTTACTATATCGTCAATTAGGTAAACTCAATTCCACATATATCGAAGGATTATTAAAAGAGATTCATGAAGATGATGGGAAGATCCATACCCGATATCAACAAGCCCTAACTTCAACTGGGCGTTTGAGTTCGATCAATCCAAACCTTCAAAATATACCAGTTCGTTTAGAAGAAGGTAGAAAAATACGTAAAGCCTTTGTTCCTTCACAACCGGGATGGGTAATGTTTGCGGCGGATTACTCTCAAATTGAATTGCGTGTTCTTGCCCATATGTCTGAGGATGAAAACCTGGTAGAAGCTTTTAATAATGATCTGGATATTCATACTAAAACGGCTATGGATGTATTCCATGTGGAGCAGGAAGCAGTAACGTCCGATATGCGCCGTGCTGCTAAGGCAGTTAACTTTGGGATTGTGTATGGTATTAGTGATTATGGTTTATCACAAAACCTAGATATTACTAGAAAAGAAGCGGCGACATTTATCGAGAATTATTTAAATAGCTTCCCAGGTGTAAAAGGATATATGGATGATATCGTTCAAGATGCGAAACAAACAGGCTACGTTACAACAATTTTGAATAGACGAAGATATTTGCCTGAAATAACAAGTTCTAACTTTAATCTCCGCAGTTTTGCAGAACGTACTGCTATGAATACACCAATTCAAGGGAGTGCAGCCGATATTATTAAAAAAGCAATGATCGATATGGCGGAAAGATTAATATCAGAAAATATGCAGACCAAAATGCTACTACAAGTACATGATGAATTAATTTTTGAGGCTCCACCAGAGGAAATTGCAATGCTAGAAAAAATAGTGCCAGAGGTGATGGAAAACGCTATTAAACTGATTGTACCTTTGAAAGTGGATTATGCCTTTGGTTCATCTTGGTATGACACGAAGTAGSEQ ID NOS: 6-10 are, like SEQ ID NO: 1, an amino acidsequence of a region of the finger domain of DNApolymerase I from Bacillus species C3_41*, Ureibacillusthermosphaericus, Bacillus subtilis, Bacillus smithiiand Geobacillus stearothermophilus respectively (* = Bei et al. 2005, Arch Microbiol, 186: 203-209;Genbank accession number DQ309765). SEQ ID NO: 6MRRAAKAVNFGIVYGISDYGLSQNLDITRKEA SEQ ID NO: 7MRRAAKAVNFGIIYGISDYGLSQNLDISRKEA SEQ ID NO: 8MRRQAKAVNFGIVYGISDYGLSQNLGITRKEA SEQ ID NO: 9MRRQAKAVNFGIVYGISDYGLSQNLGITRKEA SEQ ID NO: 10MRRQAKAVNFGIVYGISDYGLAQNLNISRKEASEQ ID NO: 11 is the amino acid sequence of truncatedDNA polymerase I isolated from Geobacillus stearothermophilus (Bst).SEQ ID NO: 11AKMAFTLADRVTEEMLADKAALWEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWYDAKSEQ ID NO: 12 is the amino acid sequence of truncated DNApolymerase I isolated from Ureibacillus thermosphaericus (Ubts)SEQ ID NO: 12AALSFKIVREIAEDLFTDTMAVHVELENEHYHTCNILGFGFTDGSNTFFVPTEVLQKSERLKSYFEDETKKKYMSDLKAAQCILKRHGINLRGVEFDLLLASYIVNPAISGDDVATLAKEFGYTDVRSNEAVYGKGAKWALPSEEVLAEHVCRKAFAIWSCKERVSNKLKENEQFDLYHDLELPLAVILGKMESEGIKVNISTLETMGQELEDKIAKLETEIYELAGETFNINSPKQLGVILFEKLGLPVIKKTKTGYSTAADVLEKLKSEHQIVQLILEYRTLAKLQSTYIEGLIKEVHPKDSKVHTRFMQALTSTGRLSSTDPNLQNIPIRLEEGRKIRKAFVPSHDGWLLFSADYSQIELRVLAHMSKDKNLVEAFNQGMDIHTRTAMEVFHVSQDDVTSNMRRAAKAVNFGIIYGISDYGLSQNLDISRKEAGEFIEKYFESFPGVKEYMDNIVQEAKLKGYVTTILNRRRYLPDITSKNFNLRSFAERTAMNTPIQGSAADIIKKAMLDIDARLNSEGLQAKLLLQVHDELIFEAPKEEIEKLEKIVPEVMESAILLDVPLKVDISYGETWYDAKSEQ ID NO: 13 is the codon optimised (for E. coli expression)nucleic acid sequence encoding the PB D422A mutant. SEQ ID NO: 13ACCGAAGTTGCATTTGAAATTGTGGAAGAAATCGATAGCACCATCCTGGATAAAGTTATGAGCGTTCATCTGGAAATGTATGATGGTCAGTATCATACCAGCGAACTGCTGGGTATTGCACTGAGTGATGGTGAAAAAGGTTATTTTGCACCGGCAGATATTGCCTTTCAGAGCAAAGATTTTTGTAGCTGGCTGGAAAATGCCACCAACAAAAAATACCTGGCAGATAGCAAAGCAACCCAGGCAGTTAGCCGTAAACATAATGTTAATGTTCACGGCGTGGAATTTGATCTGCTGCTGGCAGCATATATTGTTAATCCGGCAATTAGCAGCGAAGATGTTGCAGCAATTGCAAAAGAATTCGGCTATTTTAACCTGCTGACCAACGATAGCGTTTATGGTAAAGGTGCAAAAAAAACCGCACCGGAAATTGAAAAAATTGCCGAACATGCAGTTCGTAAAGCACGTGCAATTTGGGATCTGAAAGAAAAACTGGAAGTGAAACTGGAAGAGAACGAACAGTATGCCCTGTATAAAGAAATTGAACTGCCGCTGGCAAGCATTCTGGGCACCATGGAAAGTGATGGTGTTCTGGTTGATAAACAAATCCTGGTTGAAATGGGTCACGAGCTGAACATTAAACTGCGTGCAATTGAACAGGATATTTATGCACTGGCAGGCGAAACCTTTAACATTAATAGCCCGAAACAGCTGGGTGTGATCCTGTTTGAAAAAATCGGTCTGACCCCGATCAAAAAAACCAAAACCGGTTATAGCACCGCAGCAGATGTTCTGGAAAAACTGGCAAGCGAACATGAAATTATTGAGCAGATTCTGCTGTATCGTCAGCTGGGTAAACTGAATAGCACCTATATTGAAGGTCTGCTGAAAGAAATCCATGAGGATGATGGTAAAATCCATACCCGTTATCAGCAGGCACTGACCAGCACCGGTCGTCTGAGCAGCATTAATCCGAATCTGCAGAATATTCCGGTTCGTCTGGAAGAAGGTCGTAAAATTCGTAAAGCATTTGTTCCGAGCCAGCCTGGTTGGGTTATGTTTGCAGCAGATTATAGCCAGATTGAACTGCGTGTTCTGGCACATATGAGCGAAGATGAAAATCTGGTTGAAGCCTTTAACAACGATCTGGATATTCATACCAAAACCGCCATGGATGTTTTTCACGTTGAACAAGAAGCAGTTACCAGCGATATGCGTCGTGCAGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATCAGCGCTTATGGTCTGAGCCAGAATCTGGATATTACCCGTAAAGAAGCAGCCACCTTTATCGAAAACTACCTGAATAGCTTTCCGGGTGTGAAAGGCTATATGGATGATATTGTTCAGGATGCAAAACAGACCGGTTATGTTACCACCATTCTGAATCGTCGTCGTTATCTGCCGGAAATTACCAGCAGCAACTTTAATCTGCGTAGCTTTGCAGAACGTACCGCAATGAATACCCCGATTCAGGGTAGCGCAGCAGATATTATCAAAAAAGCCATGATTGATATGGCCGAACGTCTGATTAGCGAAAATATGCAGACCAAAATGCTGCTGCAGGTTCATGATGAACTGATTTTTGAAGCACCGCCTGAAGAAATTGCAATGCTGGAAAAAATTGTTCCGGAAGTGATGGAAAACGCCATTAAACTGATTGTTCCGCTGAAAGTGGATTATGCATTTGGTAGCAGTTGGTACGATACCAAATAASEQ ID NO: 14-is the codon optimised nucleic acid sequenceencoding the Bst D→A mutant. SEQ ID NO: 14GCCAAAATGGCATTTACCCTGGCAGATCGTGTTACCGAAGAAATGCTGGCAGATAAAGCAGCACTGGTTGTTGAAGTTGTGGAAGAAAATTATCATGATGCACCGATTGTTGGTATTGCCGTTGTTAATGAACATGGCCGTTTTTTTCTGCGTCCGGAAACCGCACTGGCCGATCCGCAGTTTGTTGCATGGCTGGGTGATGAAACCAAAAAAAAGAGCATGTTTGATAGCAAACGTGCAGCAGTTGCACTGAAATGGAAAGGTATTGAACTGTGCGGTGTTTCATTTGATCTGCTGCTGGCAGCATATCTGCTGGATCCGGCACAGGGTGTTGATGATGTTGCAGCAGCAGCAAAGATGAAACAGTATGAAGCAGTTCGTCCGGATGAAGCCGTTTATGGTAAAGGTGCAAAACGTGCCGTGCCGGATGAACCGGTGCTGGCCGAACATCTGGTTCGTAAAGCAGCCGCAATTTGGGAATTAGAACGTCCGTTTCTGGATGAACTGCGTCGTAATGAACAGGATCGTCTGCTGGTTGAACTGGAACAGCCGCTGAGCAGCATTCTGGCAGAAATGGAATTTGCCGGTGTTAAAGTGGATACCAAACGTCTGGAACAAATGGGTAAAGAACTGGCAGAACAGCTGGGCACCGTTGAACAGCGTATTTATGAGCTGGCAGGTCAAGAATTTAACATCAATAGCCCGAAACAACTGGGCGTGATTCTGTTTGAAAAACTGCAGCTGCCGGTTCTGAAAAAAACCAAAACCGGTTATAGCACCAGCGCAGATGTTCTGGAAAAACTGGCACCGTATCATGAAATTGTGGAAAACATTCTGCATTATCGCCAGCTGGGTAAACTGCAGAGCACCTATATTGAAGGTCTGCTGAAAGTTGTTCGTCCCGATACCAAAAAAGTGCACACCATTTTTAACCAGGCACTGACCCAGACCGGTCGTCTGAGCAGTACCGAACCGAATCTGCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGTCAGGCCTTTGTTCCGAGCGAAAGCGATTGGCTGATTTTTGCAGCAGATTATAGCCAGATTGAACTGCGCGTTCTGGCACATATTGCCGAAGATGATAATCTGATGGAAGCATTTCGTCGCGATCTGGATATTCATACCAAAACAGCCATGGATATTTTTCAGGTGAGCGAAGATGAAGTTACCCCGAATATGCGTCGTCAGGCAAAAGCAGTTAATTTTGGTATTGTGTATGGCATTAGCGCATATGGTCTGGCACAGAATCTGAATATTAGCCGTAAAGAAGCAGCCGAGTTTATCGAACGTTATTTTGAAAGTTTTCCGGGTGTGAAACGCTATATGGAAAATATTGTTCAAGAAGCCAAACAGAAAGGCTATGTTACCACACTGCTGCATCGTCGTCGTTATCTGCCGGATATTACCAGCCGTAACTTTAATGTTCGTAGCTTTGCAGAACGTATGGCAATGAATACCCCGATTCAGGGTAGCGCAGCCGATATTATCAAAAAAGCAATGATTGATCTGAACGCACGCCTGAAAGAAGAACGTCTGCAGGCACATCTGCTGTTACAGGTTCATGATGAACTGATTCTGGAAGCCCCTAAAGAAGAGATGGAACGTCTTTGTCGTCTGGTTCCGGAAGTTATGGAACAGGCAGTTACCCTGCGTGTTCCGCTGAAAGTGGATTATCATTATGGTAGCACCTGGTATGATGCCAAATAASEQ ID NO: 15-is the codon optimised nucleic acid sequenceencoding the Ubts D→A mutant. SEQ ID NO: 15GCAGCACTGAGCTTTAAAATCGTTCGTGAAATTGCAGAGGACCTGTTTACCGATACCATGGCAGTTCATGTTGAACTGGAAAACGAACATTATCACACGTGCAACATTCTTGGTTTTGGTTTTACCGATGGCAGCAACACCTTTTTTGTTCCGACCGAAGTGCTGCAGAAAAGCGAACGTCTGAAAAGCTATTTTGAGGATGAAACCAAAAAAAAGTATATGAGCGATCTGAAAGCAGCCCAGTGTATTCTGAAACGTCATGGTATTAATCTGCGTGGCGTTGAATTTGATCTGCTGCTGGCAAGCTATATTGTTAATCCGGCAATTAGCGGTGATGATGTTGCAACCCTGGCAAAAGAATTTGGCTATACCGATGTTCGTAGCAATGAAGCCGTTTATGGTAAAGGTGCAAAATGGGCACTGCCGAGCGAAGAGGTTCTGGCAGAACATGTTTGTCGTAAAGCATTTGCAATTTGGAGCTGCAAAGAACGCGTTAGCAATAAACTGAAAGAGAACGAACAGTTCGATCTGTATCATGATCTGGAACTGCCGCTGGCCGTTATTCTGGGTAAAATGGAAAGCGAAGGCATCAAAGTGAATATCAGCACCCTGGAAACCATGGGTCAAGAACTGGAAGATAAAATTGCCAAACTGGAAACCGAGATCTATGAACTGGCAGGCGAAACCTTTAACATTAATAGCCCGAAACAGCTGGGTGTGATCCTGTTTGAAAAACTGGGTCTGCCGGTTATCAAAAAAACGAAAACCGGTTATAGCACCGCAGCAGATGTTCTGGAAAAACTGAAATCAGAACATCAGATTGTGCAGCTGATTCTGGAATATCGTACCCTGGCCAAACTGCAGAGCACCTATATTGAAGGTCTGATCAAAGAAGTGCATCCGAAAGATAGCAAAGTGCATACCCGTTTTATGCAGGCACTGACCAGCACCGGTCGTCTGAGCAGCACCGATCCGAATCTGCAGAATATTCCGATTCGTCTGGAAGAAGGTCGTAAAATTCGCAAAGCCTTTGTGCCGAGCCATGATGGTTGGCTGCTGTTTAGCGCAGATTATAGCCAGATTGAACTGCGTGTTCTGGCACATATGAGCAAAGATAAAAATCTGGTGGAAGCCTTTAACCAAGGCATGGATATTCATACCCGTACCGCAATGGAAGTTTTTCATGTTAGCCAGGATGATGTGACCAGCAATATGCGTCGTGCAGCAAAAGCAGTTAATTTCGGTATTATCTATGGCATTAGCGCATATGGTCTGAGCCAGAATCTGGATATTTCACGTAAAGAAGCAGGCGAATTCATCGAGAAATACTTTGAAAGTTTTCCGGGTGTGAAAGAATATATGGACAACATTGTTCAAGAGGCCAAGCTGAAAGGTTATGTTACCACCATTCTGAATCGTCGTCGTTATCTGCCGGATATTACCAGCAAAAATTTCAATCTGCGTAGCTTTGCAGAACGTACCGCCATGAATACCCCGATTCAGGGTAGCGCAGCCGATATCATCAAAAAAGCAATGCTGGATATTGATGCCCGTCTGAATAGCGAAGGTCTGCAGGCAAAACTGCTGCTGCAGGTTCACGATGAACTGATTTTTGAAGCACCGAAAGAAGAGATCGAGAAGCTGGAAAAAATTGTTCCGGAAGTTATGGAAAGTGCCATTCTGCTGGATGTTCCGCTGAAAGTTGATATTAGCTATGGTGAAACCTGGTACGATGCCAAATAA

The invention will now be described by way of a non-limiting Examplewith reference to the following figures in which:

FIG. 1 gives the sequence of a region within the finger domain of DNApolymerase I from a number of species which may be modified inaccordance with the present invention. The key aspartic acid residue isin bold type.

FIG. 2 shows an overview of the strand-displacement activity assaysetup. F=fluorophore. Q=Quencher.

FIG. 3 shows a comparison of the strand-displacement activity at 25° C.of PB and the PB D422A mutant as well as for various commercial enzymesincluding the Klenow fragment (KF).

FIG. 4 shows the polymerase activity of wild type and mutant PBpolymerase at various NaCl and KCl concentrations (25° C.).

FIG. 5 is a sequence alignment of the wild type (truncated) amino acidsequences of the DNA polymerases from PB, Bst and Ubts. The large arrowindicates the 422 position where the Asp (D) is mutated to Ala (A). Thealignment is produced using Clustal X2 and is visualised using ESPript3.0 server.

FIG. 6 shows the effect of the D422A mutation on strand-displacementactivity of Bacillus stearothermophilus (large fragment) polymerase I(Bst) at 37° C. in presence of 10 mM KCl.

FIG. 7 shows the effect of the D422A mutation on strand-displacementactivity of Ureibacillus thermosphaericus (large fragment) Polymerase I(Ubts) at 37° C. in presence of 10 mM KCl.

EXAMPLES Example 1

Cloning of Sequences

PB Polymerase I Wild Type (Large Fragment) and D422A Mutant

The gene (SEQ ID NO: 3) encoding the DNA polymerase I large fragment(i.e. omitting the 5′-3′ exonuclease domain of the protein) from thePsychrobacillus sp. was cloned into the vector pET151/D-TOPO®. Thecodon-optimised variant also containing the D422A mutation (SEQ ID NO:13) was cloned into the vector pET-11a. In each case the constructencoded a His₆ tag at the N-terminus of the polymerase followed by therecognition sequence for the TEV protease, thus allowing cleavage of thetag.

Bst Polymerase I (Large Fragment) and Ubts Polymerase I (Large Fragment)and their D422A Mutant

The codon-optimized genes encoding the polymerase I large fragment fromGeobacillus stearothermophilus (Bst) and Ureibacillus thermosphaericus(Ubts, Genbank accession nr. WP_016837139) were purchased from theInvitrogen GeneArt Gene Synthesis service from Thermo Fisher Scientific.The genes (SEQ ID NOS: 14 and 15) were cloned into the vector pTrc99Aencoding an N-terminal His₆-tag by FastCloning (Li et al. (2011), BMCBiotechnology, 11:92). The corresponding mutation from Asp to Ala atposition 422 (PB polymerase I large fragment) was introduced using theQuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) andconfirmed by sequence analysis.

Protein Production and Purification

PB Polymerase I Wild Type (Large Fragment) and D422A Mutant

Recombinant protein production was performed in Rosetta 2 (DE3) cells(Novagen®). The cells grew in Terrific Broth media and gene expressionwas induced at OD_(600 nm) 1.0 by addition of 0.1 mM IPTG. Proteinproduction was carried out at 15° C. for 6-8 h. For protein purificationthe pellet of a 1-l cultivation was resuspended in 50 mM HEPES, 500 mMNaCl, 10 mM imidazole, 5% glycerol, pH 7.5, 0.15 mg/ml lysozyme, 1protease inhibitor tablet (cOmplete™, Mini, EDTA-free Protease InhibitorCocktail, Roche) and incubated on ice for 30 min. Cell disruption wasperformed by French press (1.37 kbar) and subsequently by sonicationwith the VCX 750 from Sonics® (pulse 1.0/1.0, 5 min, amplitude 25%). Inthe first step the soluble part of the His₆-tagged protein present aftercentrifugation (48384 g, 45 min, 4° C.) was purified by immobilizedNi²⁺-affinity chromatography. After a wash step with 50 mM HEPES, 500 mMNaCl, 50 mM imidazole, 5% glycerol, pH 7.5 the protein was eluted at animidazole concentration of 250 mM and further transferred into 50 mMHEPES, 500 mM NaCl, 10 mM MgCl₂, 5% glycerol, pH 7.5 by use of adesalting column.

The second step was cleavage of the tag by TEV protease performed overnight at 4° C. in 50 mM Tris pH 8.0, 0.5 mM EDTA and 1 mM DTT. Toseparate the protein from the His₆-tag and the His₆-tagged TEV proteasea second Ni²⁺-affinity chromatography has been performed in the thirdstep by applying 50 mM HEPES, 500 mM NaCl, 5% glycerol, pH 7.5. Fourthand final step of the protein purification was size-exclusionchromatography on a HiLoad 16/600 Superdex 200 pg (GE Healthcare) in 50mM HEPES, 500 mM NaCl, 5% glycerol, pH 7.5. The final protein solutionwas concentrated and stored with 50% glycerol at −20° C.

Bst Polymerase I and Ubts Polymerase I (Lame Fragment) and their D422AMutants

Recombinant protein production for Bst and Ubts polymerase I (largefragment) and their D422A mutant was performed in Rosetta 2 (DE3) cells(Novagen®). Cells grew in Luria Bertani media at 37° C. and geneexpression was induced at 0D600 nm 0.5 by addition of 0.5 mM IPTG.Protein production was carried out at 37° C. for 4 h. For proteinpurification the pellet of a 0.5-l cultivation was resuspended in 50 mMTris pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM imidazole, 0.15mg/ml lysozyme, 1 protease inhibitor tablet (cOmplete™, Mini, EDTA-freeProtease Inhibitor Cocktail, Roche) and incubated on ice for 30 min.Cell disruption was performed by sonication with the VCX 750 fromSonics® (pulse 1.0/1.0, 15 min, amplitude 25%). The soluble part of theHis₆-tagged protein present after centrifugation (48384 g, 45 min, 4°C.) was purified by immobilized Ni²⁺-affinity chromatography. After awash step with 50 mM Tris pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 10mM imidazole the protein was elution with gradually increasing theimidazole to 500 mM. Fractions containing the protein were collected andbuffer exchange was performed into 20 mM Tris pH 7.1, 100 mM KCl, 2 mMDTT, 0.2 mM EDTA and 0.2 Triton X-100 by desalting. The final proteinsolution was concentrated and stored with 50% glycerol at −20° C.

Activity Measurements

Polymerase Activity

The polymerase activity assay is based on a molecular beacon assay(modified from Summerer (2008), Methods Mol. Biol.; 429: 225-235). Themolecular beacon template consists of a 23mer loop that is connected bya GC-rich 8mer stem region (sequence is indicated in italics) and a43mer 3′ extension. Due to the stem-loop structure the FAM (donor) andDabcyl (acceptor, non-fluorescent quencher) molecules are in closeproximity and thus the FAM fluorescence signal is quenched. Upon primerextension by the DNA polymerase the stem is opened and the increase indistance of the two dyes is measured by the restoration of FAMfluorescence as relative fluorescence units in appropriate timeintervals by exciting at 485 nm and recording emission at 518 nm. Themeasurement was performed in a SpectraMax® M2^(e) Microplate Reader(Molecular Devices).

molecular beacon template (SEQ ID NO: 16) 5′-GGCCCGT^(Dabcyl)AGGAGGAAAGGACATCTTCTAGCAT ^(FAM) ACGGGCCGTCAAGTTCATG GCCAGTCAAGTCGTCAGAAATTTCGCACCAC-3′ primer (SEQ ID NO: 17)5′-GTGGTGCGAAATTTCTGAC-3′

The molecular beacon substrate was produced by incubating 20 μl of 10 μMmolecular beacon template and 15 μM primer in 10 mM Tris-HCl pH 8.0, 100mM NaCl for 5 min at 95° C. The reaction was then let to cool down atroom temperature for 2 h. The substrate solution was stored at −20° C.with a final concentration of 10 μM.

Assay Set-Up for Analyzing Effect of Different [Salt] on PolymeraseActivity of PB and PB D422A

Fifty microliter reactions consisted of 200 nM substrate and 200 μM dNTP(equimolar amounts of dATP, dGTP, dCTP and dTTP). The reaction furthercontained 5 mM MgCl₂ in 50 mM BIS-Tris propane at pH 8.5, 1 mM DTT, 0.2mg/ml BSA and 2% glycerol. Final salt concentration in the reactionbuffer has been adjusted to 25 mM, 40 mM, 60 mM, 80 mM, 110 mM, 160 mMand 210 mM NaCl or KCl for PB and 20 mM, 40 mM, 60 mM, 80 mM, 100 mM,150 mM and 200 mM NaCl or KCl for PB D422A. The activity assay wascarried out at 25° C. in black 96-well fluorescence assay plates(Corning®). The reaction was initiated by addition of protein solution,i.e. addition of polymerase.

Results are shown in FIG. 4 .

Assay Set-Up for Analyzing Specific Polymerase Activity of PB and PBD422A at 100 mM, 150 mM and 200 mM NaCl

Fifty microliter reactions consisted of 200 nM substrate and 200 μM dNTP(equimolar amounts of dATP, dGTP, dCTP and dTTP). The reaction furthercontained 5 mM MgCl₂ in 50 mM BIS-Tris propane at pH 8.5, 1 mM DTT, 0.2mg/ml BSA and 2% glycerol. Final salt concentration in the reactionbuffer has been adjusted to 100 mM, 150 mM and 200 mM NaCl,respectively. The assay was carried out at 25° C. in black 96-wellfluorescence assay plates (Corning®). The reaction was initiated byaddition of protein solution, i.e. addition of polymerase.

Results are shown in Table 3 (at end of Example).

Strand-Displacement Activity Assay

An overview of the assay setup is shown in FIG. 2 . The assay is basedon an increase in fluorescence signal that is measured upon displacementof the quenched reporter strand which is only achievable throughstrand-displacement activity of the DNA polymerase.

The substrate for the strand-displacement activity assay consists of a“cold” primer of 19 oligonucleotides (SEQ ID NO:18) and a reporterstrand consisting of 20 oligonucleotides that is labeled with the TAM RAfluorophore (F) at its 3′ end (SEQ ID NO:19). The template strandconsists of 40 oligonucleotides and is labeled with the Black HoleQuencher 2 (BHQ2) at its 5′ end (SEQ ID NO:20). The primers are annealedto the template strand leaving a one nucleotide gap at position 20 onthe template strand. The labels are in close proximity and thus thefluorophore TAM RA is quenched by BHQ2. Upon strand-displacementactivity of the DNA polymerase I the TAM RA labeled oligonucleotide isdisplaced from the template strand. As a consequence the fluorophore andthe quencher are no longer in close proximity and an increase in TAM RAfluorescence can be measured as relative fluorescence units inappropriate time intervals (excitation 525 nm, emission 598 nm,SpectraMax® M2^(e) Microplate Reader (Molecular Devices)).

5′-TATCCACCAATACTACCCT CGATACTTTGTCCACTCAAT [TAMRA]-3′3′-ATAGGTGGTTATGATGGGATGCTATGAAACAGGTGAGTTA [BHQ2]-5′

The substrate for the strand-displacement activity assay was produced byincubating 20 μl of 10 μM “cold” primer, 10 μM reporter strand and 10 μMtemplate strand in 10 mM Tris-HCl pH 8.0, 100 mM NaCl at 95° C. for 5min. The reaction was then let to cool down at room temperature for 2 h.The substrate solution was stored at −20° C. with a final concentrationof 10 μM.

Assay Set-Up for Comparison of the Specific Strand-Displacement Activityof PB, PB D422A and Commercially Known Polymerases

Fifty microliter reactions consisted of 200 nM substrate and 200 μM dNTP(equimolar amounts of dATP, dGTP, dCTP and dTTP). For PB polymerase Ithe reaction further contained 5 mM MgCl₂ in 50 mM BIS-TRIS propane atpH 8.5, 100 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA and 2% glycerol. For thecommercially known polymerase Is the respective reaction buffer suppliedby New England Biolabs have been used. Final salt concentration in thereaction buffer has been adjusted to 100 mM according to the optimalsalt for the respective polymerases. The activity assay was carried outat 25° C. in black 96-well fluorescence assay plates (Corning®). Thereaction was initiated by addition of protein solution (i.e. addition ofpolymerase).

Results are shown in FIG. 3 .

Assay Set-Up for Specific Strand-Displacement Activity of PB and PBD422A at 100 mM, 150 mM and 200 mM NaCl

Fifty microliter reactions consisted of 200 nM substrate and 200 μM dNTP(equimolar amounts of dATP, dGTP, dCTP and dTTP). The reaction furthercontained 5 mM MgCl₂ in 50 mM BIS-Tris propane at pH 8.5, 1 mM DTT, 0.2mg/ml BSA and 2% glycerol. Final salt concentration in the reactionbuffer has been adjusted to 100 mM, 150 mM and 200 mM NaCl,respectively. The assay was carried out at 25° C. in black 96-wellfluorescence assay plates (Corning®). The reaction was initiated byaddition of protein solution, i.e. addition of polymerase.

Results are shown in Table 2 below.

Assay Set-Up for Analyzing Strand-Displacement Activity of Bst/BstD422Aand Ubts/UbtsD422A

Fifty microliter reactions consisted of 200 nM substrate and 200 μM dNTP(equimolar amounts of dATP, dGTP, dCTP and dTTP). The reaction furthercontained 20 mM Tris pH 7.9 (at 25°), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mMMgSO4, 0.1% Triton X-100.

The assay was carried out at 37° C. in black 96-well fluorescence assayplates (Corning®). The reaction was initiated by addition of proteinsolution (20 ng for Bst and BstD422A, 100 ng for Ubts and UbtsD422A),i.e. addition of polymerase. For determination of the specificstrand-displacement activity (mRFU/min/μg) at a higher KCl the finalconcentration has been set to 150 mM KCl. The increase in TAMRAfluorescence was measured as relative fluorescence units in appropriatetime intervals by exciting at 525 nm and recording emission at 598 nm.The measurement was performed in a SpectraMax® M2^(e) Microplate Reader(Molecular Devices).

Results based on this strand-displacement activity assay are shown inFIGS. 6 and 7 and Table 4 below. The mutant enzymes all show enhancedactivity.

Tables

TABLE 1 Summary of different enzymatic properties for wtPB and the D422Amutant (at 25° C.). Strand- displacement Polymerase NaCl activityactivity KCl Variant (100 mM NaCl) (100 mM NaCl) T_(m) MgCl₂ (>80%activity) pH PB 310% 120% 44.8° C. 4-6 mM 25-200 mM 8.5 D422A 40-200 mMPB pol I 100% 100% 44.8° C. 3-8 mM 25-125 mM 8.5 wild type 25-115 mM

TABLE 2 Strand-displacement activity of PB D422A mutant compared to wtPBin presence of 100-200 mM NaCl. Strand-displacement activity Activity[mRFU/min/μg] NaCl wtPB PBD422A Ratio (PB D422A/wtPB) 100 9,35E+0428.8E+04 3,1 150 7,54E+04 20.5E+04 2,7 200 4,65E+04 18.1E+04 3,9

TABLE 3 DNA polymerase activity of PB D422A mutant compared to wt PB inpresence of 100-200 mM NaCl. Polymerase activity Activity [mRFU/min/μg]NaCl [mM] wtPB PBD422A Ratio (PB D422A/wtPB) 100 l,42E+06 1,66E+06 1,2150 l,02E+06 1,50E+06 1,5 200 0,55E+06 l,37E+06 2,5

TABLE 4 Strand-displacement activity of the D422A mutants of Bst andUbts compared to wt enzymes in presence of 150 mM KCl. SDA (mRFU/min/μg)Ratio SDA (mRFU/min/μg) Ratio Bst (wt) BstD422A BstD422A/Bst Ubts (wt)UbtsD422A UbtsD422A/Ubts 3,52E+05 6,99E+05 2,0 0,59E+05 2,14E+05 3,6

Example 2

Further Psychrobacillus sp. (PB) DNA polymerase mutants were also madeand tested:

Site-Directed Mutagenesis

The corresponding mutation from Asp to Ser, Lys, Val, Leu and Asn,respectively, at position 422 was introduced using the QuikChange IISite-Directed Mutagenesis Kit (Agilent Technologies).

D422V and D422L (hydrophobic residues of different lengths),

D422S (small hydrophilic),

D422N (larger hydrophilic) and

D422K (positively charged).

The starting point was the plasmid DNA of the D422A mutant. Mutationswere confirmed by sequencing analysis.

Protein production and protein purification Recombinant proteinproduction was performed in Rosetta 2 (DE3) cells (Novagen®). The cellsgrew in Terrific Broth media and gene expression was induced atOD_(600 nm) 1.0 by addition of 0.1 mM IPTG. Protein production wascarried out at 15° C. for 6-8 h. For protein purification the pellet ofa 50-ml cultivation was resuspended in 1 ml 50 mM HEPES, 500 mM NaCl, 10mM imidazole, 5% glycerol, pH 7.5, 0.15 mg/ml lysozyme and incubated onice for 20 min. Cell disruption was performed by sonication with the VCX750 from Sonics® (pulse 1.0/1.0, 1 min, amplitude 20%).

The soluble part of the His₆-tagged protein present after centrifugation(16000 g, 30 min, 4° C.) was purified with PureProteome™ Magnetic Beads(Millipore) and eluted in 50 μl 50 mM HEPES, 500 mM NaCl, 500 mMimidazole, 5% glycerol, pH 7.5.

The strand-displacement assay was performed as described in Example 1.

All these other mutants performed better in assays of stranddisplacement activity (data not shown) as compared to the wt PBpolymerase, but not as well as the PB D422A mutant.

1. A DNA polymerase comprising the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine.
 2. The DNA polymerase according to claim 1, wherein said DNA polymerase comprises an amino acid sequence which is at least 80% identical to SEQ ID NO:11, wherein the aspartic acid residue at position 422 of SEQ ID NO:11, or the equivalent aspartic acid residue in the variant sequence, is replaced by alanine.
 3. The DNA polymerase according to claim 1, wherein said DNA polymerase comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11, wherein the aspartic acid residue at position 422 of SEQ ID NO: 11, or the equivalent aspartic acid residue at the equivalent position in the variant sequence, is replaced by alanine.
 4. The DNA polymerase according to claim 1, wherein said DNA polymerase has at least 30% greater strand displacement activity as compared to a DNA polymerase with SEQ ID NO:11 but with aspartic acid at position 422, relative to SEQ ID NO:11, or at the equivalent position in the variant sequence.
 5. The DNA polymerase according to claim 1, wherein across a concentration range from 20 mM to 200 mM of NaCl, KCl, or a mixture thereof, said DNA polymerase exhibits at least 40% of its maximum polymerase activity.
 6. A composition, comprising: the DNA polymerase according to claim 1, and a buffer.
 7. A nucleic acid molecule, comprising: a nucleotide sequence encoding the DNA polymerase according to claim
 1. 8. The nucleic acid molecule of claim 7, wherein the nucleotide sequence has at least 70% sequence identity to SEQ ID NO:
 14. 9. An expression vector comprising the nucleic acid molecule of claim 8, and one or more regulatory sequences enabling transcription and translation of a protein encoded by said nucleic acid molecule.
 10. A host cell or virus, comprising one or more expression vectors according to claim
 9. 11. A host cell or virus, comprising one or more nucleic acid molecules according to claim
 8. 12. A method of producing a DNA polymerase, which comprises: (i) culturing a host cell in a growth medium, wherein the host cell comprises one or more recombinant expression vectors or one or more nucleic acid molecules encoding the DNA polymerase, under conditions suitable for expression of the encoded DNA polymerase; and optionally (ii) isolating the expressed DNA polymerase from the host cell or from the growth medium or supernatant of the growth medium, wherein the DNA polymerase comprises the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, and wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine.
 13. A method of nucleotide polymerization, said method comprising: (i) providing a reaction mixture comprising a DNA polymerase, a template nucleic acid molecule, an oligonucleotide primer which is capable of annealing to a portion of the template nucleic acid molecule and one or more species of nucleotide; and (ii) incubating said reaction mixture under conditions whereby the oligonucleotide primer anneals to the template nucleic acid molecule and said DNA polymerase extends said oligonucleotide primer by polymerizing one or more nucleotides, wherein the DNA polymerase comprises the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, and wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine.
 14. The method of claim 13, wherein said method is performed at a constant temperature.
 15. The method of claim 14, wherein said constant temperature is from 0° C. to 42° C.
 16. The method of claim 14, wherein said constant temperature is from 10° C. to 25° C. 